PROJECT SUMMARY Nicotine and alcohol dependence represent major global health concerns as leading causes of preventable morbidity and mortality. Despite their global burden, pharmacological treatment options are limited, in large part because their molecular mechanisms remain unclear. Cigarette smoking and alcohol use traits are heritable, and genome-wide association studies (GWAS) have provided robust evidence for common variants in nearly 400 genomic regions that influence smoking and/or alcohol use traits. However, the vast majority of associated variants reside in non-coding DNA, and their target genes, gene networks, and relevant neurobiological mechanisms are poorly understood. The critical first step of the proposal is to identify neurobiologically-relevant target genes of known loci by applying H-MAGMA to a comprehensive set of large, well-powered GWAS for smoking and alcohol use traits, including nicotine dependence (ND), cigarettes per day (CPD), smoking cessation (CS), alcohol use disorder (AUD), and alcohol drinks per week (DPW). Once the putative target genes are identified, we will characterize their (1) molecular function and biological pathways using gene set enrichment analyses, (2) cellular expression profiles based on single cell transcriptomic datasets, and (3) developmental expression trajectories based on temporal transcriptomic atlas. This will refine the molecular mechanism, central cell type, and developmental window critical for using and developing addiction to cigarette smoking and alcohol. Further, because we will use a coherent framework (H-MAGMA) to decipher biological underpinnings of all five GWAS of smoking and alcohol use traits, our proposed study will allow the systematic characterization and comparison of different GWAS. Our comparison will result in the identification of pleiotropic genes associated with developing addiction to cigarette and alcohol, which would reveal core mechanisms underlying addiction.